PROJECT SUMMARY The differentiation of muscle precursor cells into contractile skeletal muscle fibers is necessary for normal muscle development and regeneration and, when defective, leads to musculoskeletal diseases such as muscular dystrophies and muscle tumors. The skeletal muscle differentiation program is controlled by transcription factors of the basic helix-loop-helix family. One of these factors, Myod, initiates skeletal muscle differentiation by sequentially activating groups of genes, including those encoding muscle contractile proteins. The long term goal of this proposal is to elucidate how muscle regulatory factors like Myod activate their transcriptional targets in a temporally and spatially controlled manner to drive differentiation. The specific hypothesis of this proposal is that Pbx homeodomain proteins direct Myod to a subset of its transcriptional targets, thereby regulating the competence of muscle precursor cells to differentiate. Two findings support this hypothesis: 1. Pbx proteins bind in a complex with Myod on the Myogenin promoter. 2. Pbx proteins are required for Myod to activate Myogenin expression and the fast- twitch muscle-specific differentiation program in zebrafish embryos. These findings present a unique opportunity to use skeletal muscle development to determine how homeodomain proteins direct specific cellular differentiation programs. Understanding how Pbx and Myod factors interact may ultimately be useful in improving the control over muscle precursor differentiation, with implications for treating musculoskeletal diseases. This proposal will utilize a combination of approaches in mouse and zebrafish embryos as well as in mammalian cell culture to allow a comprehensive biochemical, genomic and developmental genetic analysis of how Pbx regulates skeletal muscle differentiation and Myod activity. The Specific Aims of this proposal are: Aim 1. Test whether Pbx proteins are required for a subset of gene expression during mammalian skeletal muscle differentiation. Skeletal muscle development will be analyzed in Pbx-deficient mouse embryos to determine which mouse Pbx genes are required for Myogenin expression and fast- or slow-twitch muscle differentiation. To comprehensively determine the subset of mammalian Pbx-dependent muscle gene expression, expression array analyses on RNA from Pbx knockout mouse embryos will be used. Aim 2. Distinguish between an instructive or permissive mechanism for Pbx regulation of Myod targets. To test whether the Pbx binding site in the Myogenin promoter is required for Myod binding and chromatin modifications, reporter assays in mouse cell lines will be used. Genetic interaction experiments in zebrafish embryos will determine whether Pbx proteins are instructive or permissive in overcoming the activity of the fast-muscle repressor Prdm1. To test whether Pbx proteins bind broadly to, or specifically to only a subset of, muscle gene promoters, genome-wide Pbx binding sites will be determined using a global chromatin immunoprecipitation approach. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE The differentiation of muscle precursor cells into contractile skeletal muscle fibers is necessary for normal muscle development and regeneration and, when defective, leads to musculoskeletal diseases such as muscular dystrophies and muscle tumors. This proposal will identify molecular mechanisms that drive muscle precursor cells into differentiated muscle fibers. Understanding these mechanisms ultimately may be useful in improving the ability to control muscle precursor differentiation and, therefore, relevant to developing therapies for treating musculoskeletal diseases.